Probe for visualizing phosphorylation/dephosphorylation of protein and method of detecting and quantifying phosphorylation/dephosphorylation of protein

ABSTRACT

As a versatile method of detecting and assaying intracellular protein phosphorylation and dephosphorylation that enables nondestructive monitoring as well as spatial and temporal analysis for living cells, animal bodies, plant bodies and the like, a probe for imaging protein phosphorylation and dephosphorylation, which comprises a tandem fusion unit composed of a substrate domain that contains a phosphorylation and dephosphorylation site, a linker sequence and a phosphorylation recognition domain, interposed between a donor chromophore and an acceptor chromophore that cause fluorescence resonance energy transfer, is used.

This application is a 371 of PCT/JP01/02360, filed Mar. 23, 2001.

TECHNICAL FIELD

The present invention relates to a probe for detecting and assayingprotein phosphorylation and dephosphorylation. More particularly, thepresent invention relates to a probe for imaging protein phosphorylationand dephosphorylation comprising a substrate domain that contains a sitethat can be phosphorylated and a phosphorylation recognition domain,bound together by a linker sequence, interposed between a donorchromophore and an acceptor chromophore that cause fluorescenceresonance energy transfer to occur, as well as a method for detectingand assaying protein phosphorylation and dephosphorylation using thesame.

BACKGROUND ART

Protein phosphorylation by intracellular kinases is one of the mostcritical reactions in signaling within cells and is known to playimportant roles in various processes such as survival, proliferation,and differentiation of cells (Cell 100, 113-127 (2000)). Protein kinasescatalyze transfer of the γ-phosphate of ATP and phosphorylation ofhydroxyl groups of serines, threonines and/or tyrosines on the substrateproteins, and upon such phosphorylation, substrate proteins are subjectto conformational changes due to negative charges of the phosphates,which subsequently triggers their enzymatic activation and interactionwith their respective target proteins. Therefore, it is expected that byscreening substances that enhance or suppress intracellular signalingtriggered by protein phosphorylation and dephosphorylation, not only maydiagnosis of diseases become possible, but important information for thedevelopment of new drugs may be obtained, as well.

Conventionally, analysis of signaling related to the kinase proteins hasbeen preformed using means such as electrophoresis, immunocytochemistry,and kinase assay in vitro. However, these conventional methods aredestructive methods and could not provide information on spatial andtemporal analysis of signals from protein phosphorylation anddephosphorylation in living cells.

In contrast, unlike kinase signaling, second messenger signaling such asCa²⁺ (Nature 388, 882-887 (1997), inositol 1,4,5-triphosphate (Science248, 1527-1530 (1999)), diacylglycerol (J. Cell Biol. 140, 485-498(1998)), cyclic AMP (Nature 349, 694-697 (1997); Nat. Cell Biol. 2,25-29 (1999)) and cyclic GMP (Anal. Chem. 72, 5918-5924 (2000)) has beenvisualized using fluorescent indicators; it has been reported that insuch measurement methods, highly accurate spatial and temporal analysisof second messenger signaling in single living cells is made possible(Curr. Opinion Neurobiol. 10, 416-421 (2000)).

In recent years, along with probes for visualizing second messengersignaling, probes for visualizing kinase signaling in living cells havebeen studied and a few have been reported (Anal. Biochem. 195, 148-152(1991); NeuroReport 7, 2695-2700 (1996); FEBS Lett. 414, 55-60 (1997);Nat. Biotechnol. 18, 313-316 (2000)). However, these imaging probes areall based on conformational changes of the substrate peptides themselvesupon phosphorylation. Because controlling such conformational changes isimpossible, such probes were only applicable to specific kinasesignaling and lacked practicality.

Accordingly, the invention of the present patent application has beenmade in view of the above problems, and the object of the presentinvention is to provide a practical method for the detection andmeasurement of protein phosphorylation and dephosphorylation in livingcells, animal bodies, plant bodies etc., that enables a non-destructivemethod for monitoring and further enables spatial and temporal analysis,thereby solving the problems of conventional techniques.

DISCLOSURE OF INVENTION

In order to solve the above-described problems, the present inventionfirstly provides a probe for imaging protein phosphorylation anddephosphorylation, which comprises a substrate domain that contains asite that can be phosphorylated and a phosphorylation recognitiondomain, bound together by a linker sequence, interposed between a donorchromophore and an acceptor chromophore that cause fluorescenceresonance energy transfer to occur.

The present invention provides, secondly, the above probe for imagingprotein phosphorylation and dephosphorylation, wherein the donorchromophore and the acceptor chromophore are fluorescent proteins eachhaving different fluorescence wavelengths; thirdly, the above probe forimaging protein phosphorylation and dephosphorylation, wherein the donorchromophore and the acceptor chromophore that cause fluorescenceresonance energy transfer to occur, are different color mutants of agreen fluorescent protein; and fourthly, the above probe for imagingprotein phosphorylation and dephosphorylation, wherein the mutants ofthe green fluorescent protein are a cyan fluorescent protein and ayellow fluorescent protein.

Furthermore, the present invention fifthly provides the above-describedprobe for imaging protein phosphorylation and dephosphorylation, whereinthe site that can be phosphorylated in the substrate domain contains anamino acid residue selected from tyrosine, serine and threonine.

The present invention provides, sixthly, the probe for imaging proteinphosphorylation and dephosphorylation, wherein the phosphorylationrecognition domain is an endogenous domain selected from SH2 domain,phosphotyrosine binding domain or WW domain; further, seventhly, theprobe for imaging protein phosphorylation and dephosphorylation, whereinthe phosphorylation recognition domain is a single chain antibodyobtained using the phosphorylated substrate domain as an antigen.

Further, the present invention eighthly provides any one of the aboveprobe for imaging protein phosphorylation and dephosphorylation, whichcomprises a localization sequence at the terminal end.

Also, the present invention provides, ninthly, a method for screeningsubstances that enhance or suppress protein phosphorylation, whichcomprises making the probe for imaging protein phosphorylation anddephosphorylation of any one of the first to eighth inventions coexistwith a candidate substance, and measuring the change in efficiency offluorescence resonance energy transfer (FRET) before and after additionof the candidate substance.

Further, tenthly, the present invention provides a method for screeningsubstances that enhance or suppress protein phosphorylation, whichcomprises making the probe for imaging protein phosphorylation anddephosphorylation of any one of the first to eighth inventions, whereinthe substrate domain has been phosphorylated, coexist with a candidatesubstance, and measuring the change in FRET efficiency before and afteraddition of the candidate substance.

The present invention eleventhly provides, the above method forscreening substances that enhance or suppress protein phosphorylation,wherein the probe and che candidate substance are made to coexist byintroducing the probe for imaging protein phosphorylation anddephosphorylation in to cells.

Still further, twelfthly, the present invention provides a method forassaying a substance that causes protein phosphorylation, whichcomprises introducing the probe for imaging protein phosphorylation anddephosphorylation of any of the first to eighth inventions into cells,and measuring the change in FRET efficiency before and after addition ofthe candidate substance.

And, thirteenthly, the present invention also provides a method forassaying a substance that causes protein dephosphorylation, whichcomprises introducing the probe for imaging protein phosphorylation anddephosphorylation of any one of the first to eighth inventions, whereinthe substrate domain has been phosphorylated, and measuring the changein FRET efficiency before and after addition of the candidate substance.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a scherntatic diagram that describes the construction andprinciple of the probe for imaging protein phosphorylation anddephosphorylation of the present invention 1 to 8 represent thefollowing: (1 a: probe for imaging protein phosphorylation anddephosphorylation (before phosphorylation), 1 b: probe for imagingprotein phosphorylation and dephosphorylation (after phosphorylation),11: tandem fusion unit, 2 a: substrate domain (before phosphorylation),2 b: substrate domain (after phosphorylation) 21 a: phosphorylation site(before phosphorylation), 21 b: phosphorylation site (afterphosphorylation). 3: phosphorylation recognition site, 4: linkersequence, 5: donor chromophore, 5′: acceptor chromophore, 6:phosphorylation substance, 7: dephosphorylation substance)

FIG. 2 is a schematic representation that shows specific structures ofthe probe for imaging protein phosphorylation and dephosphorylationconstructed as an Example of the present invention.

FIG. 3 shows the result of immunoblotting using a phosphotyrosineantibody, when the probe for imaging protein phosphorylation anddephosphorylation of FIGS. 2( a) and 2(b) were introduced into cells, asdescribed in the Example of the present invention.

FIG. 4A is a fluorescence image of CFP after introducing the probe forimaging protein phosphorylation and dephosphorylation of FIG. 2( b) intocells, taken using an emission filter for CFP (480 nm±15 nm).

FIG. 4B is a representation of the pseudocolor images that shows timecourse change of emission ratios between CFP (480±15 nm) and YFP(535±12.5 nm) (herein after referred to as CFP/YFP) excited at 440+10nm, when cells to which the probe for imaging protein phosphorylationand dephosphorylation of FIG. 2( b) were introduced were stimulated withinsulin.

FIG. 4C is a graph that shows the time course change in CFP/YFP in thecytosol and the nucleus when excited at 440±10 nm, when the cells towhich the probe for imaging protein phosphorylation anddephosphorylation of FIG. 2( b) were introduced were stimulated withinsulin.

FIG. 4D is a graph that shows the time course change of CFP/YFP(excitation at 440±10 nm) in the cytosols of the cells to which theprobe for imaging protein phosphorylation and dephosphorylation of FIG.2( b) were introduced and treated with tyrphostin (an inhibitor ofinsulin receptor) (▪), and the cells to which the probe for imagingprotein phosphorylation and dephosphorylation of FIG. 2( c) wereintroduced (□), when stimulated with insulin.

FIG. 5A is a representation of the pseudocolor images that shows thetime course change of CFP/YFP (excitation at 440±10 nm) in the nucleusand the cytosol of the cells to which the probe for imaging proteinphosphorylation and dephosphorylation that contains a nuclear-exportsignal sequence as shown in FIG. 2( d) were introduced, afterstimulation with insulin.

FIG. 5B is a graph that shows the time course change of CFP/YFP(excitation at 40±10 nm) in the cytosol when the cells to which theprobe for imaging protein phosphorylation and dephosphorylation of FIG.2( d) were introduced were stimulated with insulin of variousconcentration.

FIG. 6A is a graph that shows the time course change of CFP/YFP(excitation at 40±10 nm) in the cytosol when the cells to which theprobe for imaging protein phosphorylation and dephosphorylation of FIG.2( b) and FIG. 2( e) were introduced were stimulated with insulin.

FIG. 6B is a representation of the confocal laser fluorescence imagethat confirms the co-localization of the probe for imaging proteinphosphorylation and dephosphorylation of FIG. 2( e) and the insulinreceptor in the cells to which the probe for imaging proteinphosphorylation and dephosphorylation of FIG. 2( e) were introduced,when stimulated with insulin.

BEST MODE FOR CARRYING OUT THE INVENTION

As described above, protein phosphorylation by intracellular kinases isone of the most important steps in intracellular signaling andparticipates profoundly in processes such as survival, proliferation,and differentiation of cells. Accordingly, protein phosphorylation anddephosphorylation are observed as a phenomenon related to the cause orcertain symptoms of various diseases. In other words, if a method fordetecting and assaying phosphorylation of specific proteins is realized,early diagnosis of various diseases may become possible. Moreover, therealization of a method for screening factors or substances that enhanceor inhibit protein phosphorylation and dephosphorylation may contributegreatly to the discovery of substances relating to such diseases and thedevelopment of novel treatment drugs.

The probe for imaging protein phosphorylation and dephosphorylation ofthe present invention is a probe that makes visible and thereby enablesthe detection and assay of phosphorylation of protein by phosphorylatingsubstances. The structure and principle of such probes for proteinphosphorylation and dephosphorylation is shown in FIG. 1.

Specifically, the probe for imaging protein phosphorylation anddephosphorylation (1 a) of the present invention comprises a tandemfusion unit (11) that comprises a substrate domain (2 a), which containsa site that can be phosphorylated (21 a) and a phosphorylationrecognition domain (3) bound together by a linker sequence (4),interposed or sandwiched between a donor chromophore (5) and an acceptorchromophore (5′) that cause fluorescence resonance energy transfer tooccur.

For the probe for imaging protein phosphorylation and dephosphorylation(1 a) of the present invention, for example, when the phosphorylationsite (21 a) of the substrate domain (2 a) is phosphorylated by aphosphorylation substance (6), the adjacent phosphorylation recognitiondomain (3) recognizes it, and specifically interacts with thephosphorylated substrate domain (21 a). In the probe for imaging proteinphosphorylation and dephosphorylation (1 b) wherein such an interactionoccurred, the donor chromophore (5) and the acceptor chromophore (5′)come into close proximity to each other; therefore, upon exposure toexternal light, excitation of the donor chromophore (5) followed by anenergy transfer to the acceptor chromophore takes place, resulting in achange in efficiency of fluorescence resonance energy transfer (FRET).Thus, by detecting such change in FRET, phosphorylation (2 b) of thesubstrate domain (2 a) can be confirmed.

In a similar manner, when the probe for imaging protein phosphorylationand dephosphorylation (1 b) containing a phosphorylated substrate domain(21 b) coexists with a dephosphorylation substance (7), and thephosphorylated site of the substrate domain (2 b) is dephosphorylated(21 a), then interaction between the phosphorylation recognition domain(3) and the substrate domain (2 b) disappears, which leads to theseparation of the donor chromophore (5) and the acceptor chromophore(5′). By exposing external light, only the excitation of the donorchromophore (5) occur without energy transfer to the acceptorchromophore. Accordingly, dephosphorylation of the substrate domain (2b) can be detected from the change that appears in the FRET efficiency,using fluorescence analysis.

Each unit constituting the probe for imaging protein phosphorylation anddephosphorylation of the present invention is described morespecifically. First, as described above, the tandem fusion unit (11)consists of the substrate domain (2 a), the phosphorylation recognitiondomain (3) and the linker sequence (4) that bind them together. Here,the sequence or structure of the substrate domain (2 a) is notrestricted as long as it contains a site that can be phosphorylated (21a). The site capable of being phosphorylated (21 a) usually contain isan —OH group, and natural amino acids such as tyrosine (Tyr) serine(Ser), threonine (Thr) and the like, as well as peptides to which an OHgroup is introduced by chemical modification may be exemplified.

Next, the phosphorylation recognition domain recognizes phosphorylationof the substrate domain (2 a) and interacts specifically with thephosphorylated substrate domain (2 b). The phosphorylation recognitiondomain may have any structure as long as it fulfills the aboveconditions. For example, endogenous domains such as SH2 domain,phosphotyrosine binding domain, WW domain and the like that are known torecognize specific phosphorylated substrates are applicable. Further,for detecting and assaying a substrate domain (2 b) for which thephosphorylation recognition domain (3) that interacts with it isunknown, a single chain antibody may be prepared using thephosphorylated substrate domain (2 b) as an antigen, and used as thephosphorylation recognition domain (3). By using such an antibody, aphosphorylation recognition domain (3) that interacts specifically withany desired phosphorylated substrate domain (2 b) can be obtained,thereby enhancing the versatility of such probe for imaging proteinphosphorylation and dephosphorylation. The single chain antibodies thatutilize the substrate domain (2 b) as an immunogen may be prepared byknown immunological means.

Next, in the probe for imaging protein phosphorylation anddephosphorylation of the present invention, the sequence and length ofthe linker sequence (4) are not limited as long as it enablesappropriate flexibility and does not contain a site that can bephosphorylated. When the sequence (4) contains a site (21 a) that can bephosphorylated, the site may be phosphorylated by a phosphorylationsubstance (6), which makes the accurate detection and assay of proteinphosphorylation impossible. The linker sequence (4) may be anypolypeptide or oligopeptide, which preferably has a chain length longenough to enable interaction between the phosphorylation recognitionsite (3) and the substrate domain (2 b) upon phosphorylation of thesubstrate domain (2 b), thereby approximating the donor chromophore (5)and the acceptor chromophore (5′).

The probe for imaging protein phosphorylation and dephosphorylation ofthe present invention comprises the tandem fusion unit (11) of theabove-described structure, interposed between a donor chromophore (5)and an acceptor chromophore (5′) that cause fluorescence resonanceenergy transfer to occur; when the substrate domain (2 a) isphosphorylated, a change in FRET is induced by the mechanism describedpreviously. Such donor chromophore (5) and acceptor chromophore (5′) maybe selected from various fluorescent substances; particularly,fluorescent proteins are considered. The chromophores may be anysubstances that show fluorescence at different wavelengths upon exposureto external light, and cyan fluorescent protein is (CFP) and a yellowfluorescent protein (YFP), which are mutants of the green fluorescentprotein (GFP), are preferable. CFP and YFP are particularly preferable;their mutated forms may be prepared in accordance with their use andutilized as the donor and/or acceptor chromophore.

In the above-described probe for imaging protein phosphorylation anddephosphorylation, the domain adjacent to the donor chromophore may beeither of the substrate domain (2 a) or the phosphorylation recognitiondomain (3). Since the preferable linking order differs depending on thestructure or steric hindrance of these domains and linker sequence (4),the order may be selected according to the combination of the substratedomain (2 a) and the phosphorylation recognition domain (3).

Further, the probe for imaging protein phosphorylation anddephosphorylation of the present invention may contain a variety oflocalization sequences at terminal end. Such localization sequences canrecognize specific cells, specific regions in the cells or specifictissues, and therefore can localize the probe for imaging proteinphosphorylation and dephosphorylation. Specifically, anuclear-export-signal sequence or a plasma membrane binding sequencesuch as pleckstrin homology (PH) domain may be ligated as a localizationsequence.

The probe for imaging protein phosphorylation and dephosphorylation ofthe present invention is as described above. But the method for itspreparation is not particularly limited, and may be constructed by totalsynthesis; however, it is preferable to ligate each domain by knowngenetic engineering techniques such as polymerase chain reaction (PCR).Here, various restriction sites and the like may also be inserted.

In the invention of the present patent application, a method forscreening substances that enhance or suppress protein phosphorylationusing the above-described probe for imaging protein phosphorylation anddephosphorylation is also provided. In other words, if the substratedomain (2 a) of the probe for imaging protein phosphorylation anddephosphorylation (1 a) is phosphorylated when the probe for imagingprotein phosphorylation and dephosphorylation of the present inventionand a candidate substance coexist, protein phosphorylation is detectedby the change in FRET under the mechanism previously described, therebyenabling the screening of substances that phosphorylate the substratedomain (2 a). These candidate substances may act directly as a proteinkinase that phosphorylate the protein, or may be substances that act atan early stage of intracellular signaling, that is, act as a proteinkinase activating substance.

On the other hand, in order to confirm enhancement or suppression ofdephosphorylation by a candidate substance and to screen a substancethat enhance or suppress dephosphorylation, the substrate domain (2 a)of the probe for imaging protein phosphorylation and dephosphorylation(1 a) is first phosphorylated (2 b), and the change in FRET that occurwhen in coexistence with a candidate substance is measured.

In the above-described method for screening substances that enhance orsuppress phosphorylation and dephosphorylation, the probe for imagingprotein phosphorylation and dephosphorylation (1 a) maybe made tocoexist with the candidate substance in a solution, for which, forexample, the pH, salt concentration or the like is adjusted, oralternatively, the probe for imaging protein phosphorylation anddephosphorylation may be introduced into cells by genetic engineeringtechniques thereby made to coexist with the candidate substances. Here,the candidate substances may be resent outside the cells or may beincorporated into the cells; further, they may be pre-introduced intothe cells by genetic engineering techniques. The candidate substancesmay also be enzymes, receptors or the like that exist in the cells.

Further, by using the probe for imaging protein phosphorylation anddephosphorylation of the present invention, the phosphorylatingsubstances may be assayed, as well. In other words, by introducing theabove probe for imaging protein phosphorylation and dephosphorylationinto the cells, and measuring the changes in FRET efficiency before andafter addition of the candidate substance, the amount of phosphorylationsubstances can be determined. For example, for substance A known tophosphorylate protein a, by observing the time course changes that occurin FRET in vitro when various concentrations of the phosphorylationsubstance A are in coexistence with the probe for imaging proteinphosphorylation and dephosphorylation, and the time at which each FRETvalue reaches a plateau is predetermined. By creating a calibrationcurve of the FRET value and the concentration of substance A at thattime, preparing the probe for imaging phosphorylation anddephosphorylation that contains protein a, which is phosphorylated bysubstance A, introducing such the probe into cells, and measuring theFRET value, substance A in the cells can be quantitated. Likewise,quantitative analysis of dephosphorylation substances may be performedin a similar manner.

As has been described previously, known genetic engineering techniquesare applicable for as a method for introducing the probe for imagingprotein phosphorylation and dephosphorylation into the cells.Specifically, an expression vector in which the probe for imagingprotein phosphorylation and dephosphorylation is incorporated may beintroduced into the cells by known methods such as electroporation, thecalcium phosphate method, the liposome method, the DEAE dextran method.Thus, by introducing the probe for imaging protein phosphorylation anddephosphorylation into the cells and making the probe coexist withphosphorylation (or dephosphorylation) substances, anin vivo method fordetecting and assaying protein phosphorylation (or dephosphorylation)that does not require the destruction of the cells is enabled.

The probe for imaging protein phosphorylation and dephosphorylation ofthe present invention is advantageous, not only for enabling the imagingof kinase signal transduction in single live cells at high spatial andtemporal resolution, but also for being valuable in multi-cell analysis,which aims for the high-throughput screening of substances that regulatephosphorylation or dephosphorylation (Science 279, 84-88 (1998); DrugDiscovery Today 4, 363-369 (1999).

Further, in the probe for imaging protein phosphorylation anddephosphorylation of the present invention, a polynucleotide for theexpression of the probe may be introduced into cells and used for theontogenesis of non-human totipotent cells, thereby creating an animal ora progeny animal in which the probe for imaging protein phosphorylationand dephosphorylation and the phosphorylation (or dephosphorylation)substance coexist in all of its cells. These so-called non-humantransgenic animals may be produced in accordance with known productionmethods (for example, Proc. Natl. Aced. Sci. USA 77, 7348-, (1980)). Thenon-human transgenic animals described above possess the probe forimaging protein phosphorylation and dephosphorylation in all of theirsomatic cells, and therefore, may be used to measure the concentrationof phosphorylation (or dephosphorylation) substances in their cells ortissues; or by introducing candidates of phosphorylation (ordephosphorylation) substances, phosphorylation (or dephosphorylation)enhancing substances, and phosphorylation (or dephosphorylation)inhibiting substances, such as drugs an toxins, into their bodies,substances that show effect in cells or tissues may be screened.

Hereinafter, the present invention is described in further detail by theExamples with reference to the accompanying drawings. Of course, itshould be needless to mention that the present invention is not limitedto the following Examples and that various modifications may be made forthe details.

EXAMPLES

Among various phosphorylation substances, nonreceptor tyrosine kinasesand seine/threonine kinases function throughout the entire signaltransduction cascades. On the other hand, tyrosine kinase receptors suchas insulin receptor and hormone receptor function at the beginning of anumber of signal transduction cascades.

In the following examples, a probe for imaging protein phosphorylationand dephosphorylation using insulin signal transduction protein wasevaluated for the detection and assay of protein phosphorylation byinsulin receptor, which is also a protein kinase.

<Preparation>

In the following examples, samples and reagents were used as follows:

Human insulin was purchased from Peptide Institute, Inc. (Osaka, Japan).

Ham's F-2 medium, fetal calf serum, Hank's balanced salt solution andLipofectAMINE 2000 reagent were obtained from Life Technologies(Rockville, Md.).

Tyrphostin 25 was purchased from Sigma Chemical Co. (St. Louis, Mo.).

Anti-phosphotyrosine antibody (PY20) andanti-β-subunit of human insulinreceptor antibody were purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, Calif.).

Anti-GFP antibody were purchased from by Clontech (Palo Alto, Calif.).

Anti-rabbit IgG antibody labeled with Cy5 was obtained from JacsonImmunoResearch Lab., Inc. (Pennsylvania, PA).

Other chemicals used were all of analytical reagent grade.

Example 1 Preparation of the Imaging Probe for the Detection of ProteinPhosphorylation by Insulin Receptor

(1) Plasmid Construction

FIG. 2 is a representation of the specific structure of each probe forimaging protein phosphorylation and dephosphorylation that wereprepared.

As shown in FIG. 2, the imaging probe was prepared so as to comprise atandem fusion unit wherein a substrate domain containing a site to bephosphorylated and a phosphorylation recognition domain are boundtogether by a linker sequence, which is interposed between two mutantsof the green fluorescent protein.

First, as a fluorescent protein, cyan fluorescent protein (CFP) andyellow fluorescent protein (YFP), which are different-colored mutants ofthe green fluorescent protein (GFP) originating from Aequorea Victoria,were used. Further, CFP was subjected to additional mutations ofF64L/S65T/Y66W/N1461/M153T/V163A/N212K, and YFP was subjected toadditional mutations of S65G/V68L/Q69K/S72A/T203Y.

Next, as a substrate domain, a tyrosine phosphorylation domain (Y941:SEQ ID NO: 1) derived from insulin receptor substrate-1 (IRS-1) wasused. In this domain, insulin receptor phosphorylates the tyrosineresidue 941 in an insulin dependent manner (Mol. Cell Biol. 13,7418-7428 (1993)).

Next, as a phosphorylation recognition domain, an N-terminal SH2 domain(SH2n: p85₃₃₀₋₄₂₉) of p85 regulatory subunit of bovinephosphatidylinositol 3-kinase, which has been reported to bind to thephosphorylation substrate domain of IRS-1 protein, was chosen. (J. Biol.Chem. 267, 25958-25966)

As a linker sequence (Ln), the oligopeptide of SEQ ID NO: 2 was used.

Restriction sites shown in FIG. 2 were inserted to the cDNAs of CFP,YFP, the substrate domain and the phosphorylation recognition domain,using standard polymerase chain reaction (PCR). All cloning enzymes werepurchased from Takara Biomedical (Tokyo, Japan). PCR fragments weresequenced using AB1310 genetic analyzer.

Further, cDNA encoding each probe for imaging protein phosphorylationand dephosphorylation was subcloned at Hind III and Xba I sites of amammalian expression vector, pcDNA3.1 (+) (Invitrogen Co., Carlsbad,Calif.).

(2) Optimization of the Structure of the Probe for Imaging ProteinPhosphorylation

In the probe for imaging protein phosphorylation and dephosphorylationshown in FIG. 2( a) to (e), the order of SH2n and Y941 in the tandemfusion units of probe (a) and probe (b) are reversed.

In the present study, to determine which tandem fusion unit, that ofprobe (a) or probe (b), is more efficiently phosphorylated by insulinreceptor, immunoblotting was performed using phosphotyrosine antibodyafter stimulating CHO-IR cells expressing probe (a) and probe (b) with100 nM insulin.

First, IR cells were cultured in 6-well plates and were transfected with2 μg of each plasmid containing probe (a) cDNA and probe (b) cDNA.CHO-HIR cells overexpressing human insulin receptor were cultured inHam's F-12 medium supplemented with 10% fetal calf serum at 37° C. in 5%CO₂. The cells were transfected with LipofectAMINE 2000 reagent. 12 to24 hours after the transfection, the cells were spread onto glass bottomdishes, glass coverslips or plastic culture dishes.

Next, CHO-IR cells expressing probe (a) and probe (b) were stimulatedwith 100 nM of insulin for 20 minutes at 25° C.

The cells were lysed with an ice-cold lysis buffer (50 mM Tis-HCl, pH7.4, 100 mM NaCl, 1 mM EDTA. 0.1% Triron X-100, 10 mM NaF, 2 mM sodiumorthovanadate, 1 mM PMSF, 10 μg/mL pepstatin, 10 μg/mL leupeptin, 10μg/mL aprotinin). The Imaging probes for protein phosphorylation wereimmunoprecipitated from the whole cell lysates of the CHO-IR cells withanti-GFP antibody for 2 hours at 4° C.

Protein G-Sepharose 4 FF beads were used to absorb theimmunoprecipitates and then washed four times with an ice-cold washingbuffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.1% TrironX-100, 10 mM NaF, 2 mM sodium orthovanadate, 1 mM PMSF, 10 μg/mLpepstatin, 10 μg/mL leupeptin, 10 μg/mL aprotinin). The samples wereseparated by SDS-polyacrylamide gel electrophoresis and analyzed by animmunoblotting method Using anti-phosphotyrosine antibody (PY20, 1:500dilution).

The result of the immunoblotting is shown in FIG. 3.

As shown in FIG. 3, probe (b) was well phosphorylated by insulinreceptor, whereas probe (a) was poorly phosphorylated. This indicatesthat in the present experiment, the tandem fusion unit linked in theorder shown in FIG. 2( b) is more effective as a probe for imagingprotein phosphorylation than the tandem fusion unit linked in the ordershown in FIG. 2( a).

In view of the x-ray crystal structure of insulin receptor in complexwith a substrate peptide derived from IRS-1, the difference in thephosphorylation efficiency between probe (a) and probe (b) may beascribed to the difference in steric effect.

In the following example, phosphorylation by insulin receptor wasdetected using probe (b).

Example 2 Detection of Phosphorylation Using the Probe for ImagingProtein Phosphorylation

The increase in FRET efficiency after the phosphorylation of probe (b)by an insulin receptor was observed.

CHO-IR cells were transfected using the cDNA encoding probe (b) insertedin a mammalian expression vector, as described in Example 1.

After serum starvation with a serum-free medium, the culture medium wasreplaced with a Hank's balanced salt solution. 3 to 5 days after thetransfection, the cells were observed at room temperature on a CarlZeiss Axiovert 135 microscope with a cooled CCD camera MicroMAX (RoperScientific Inc., Tucson, Ariz.) controlled by MetaFluor (UniversalImaging, West Chester, Pa.) in accordance with known methods (Anal.Chem. 72, 5918-5924 (1999)).

The exposure time at 440±10 nm excitation was 100 ms. The fluorescenceimages were obtained through 480±15 nm and 535±12.5 nm filters with a40× oil immersion lens (Carl Zeiss, Jena, Germany).

FIG. 4A shows fluorescence microscope images of probe (b) expressioncells, taken using an emission filter (480±15 nm) for CFP.

Probe (b) was found to be distributed uniformly in both the cytosoliccompartment and in the nucleus.

Next, to evaluate the response of probe (b) for its phosphorylation,CHO-IR cells expressing probe (b) were stimulated with 100 nM of insulinin the same manner as described in Example 1.

FIG. 4B shows the time course changes of pseudocolor images of emissionratio of CFP at 480±15 nm to that of YFP at 535±12.5 nm excited at440±10 nm.

Further, 4C shows the time courses of the emission ratio changes in thecytosol and in the nucleus. The administration of insulin caused a rapidand significant decrease in the cytosolic emission ratio for cellsexpressing probe (b), whereas the emission ratio in the nucleus showedno significant change (FIGS. 4B and 4C).

Furthermore, the insulin-induced change in emission ratio in the cytosolwas completely suppressed when the cells were pretreated with 500 μMtyrphostin, an inhibitor for insulin receptor. As a negative control,CHO-IR cells expressing probe (c), in which tyrosine was replaced withalanine at the phosphoacceptor site of the substrate domain, weresimulated with insulin; however, no significant change in the cytosolicemission ratio was observed (FIG. 4D).

The above results demonstrate that FRET from CFP to YFP increased uponphosphorylation of Y941 of probe (b) in the cytosolic compartment andsubsequent binding of the phosphorylated Y941 with the adjacentphosphorylation recognition domain SH2n. Accordingly, this resultindicates that probe (b) may be effective as a probe for imaging proteinphosphorylation by insulin receptor in single live cells.

However, no significant change in FRET efficiency was observed in thenucleus; a more rigidly packed conformation of tyrosine-phosphorylatedprobe (b), compared to the floppy conformation of unphosphorylated probe(b) due to the existence of the linker sequence, may have restricted thetraffic of the phosphorylated probe (b) through the nuclear pore, whichforced the phosphorylated probe (b) to remain in the cytosoliccompartment.

Example 3 Probe for Imaging Protein Phosphorylation containing aNuclear-export-signal Sequence

To prevent the probe for imaging protein phosphorylation from beingtransferred to the nucleus, where FRET changed did not occur uponinsulin stimulation, as described in Example 2, a probe for imagingprotein phosphorylation having a nuclear-export-signal sequence (d) wasdeveloped. AS the nuclear-export-signal sequence, anuclear-export-signal sequence (nes; SEQ ID NO: 3) derived from humanimmunodeficiency virus protein, Rev (EMBO J. 16, 5573-5581), was linkedto the terminal end of the probe for imaging protein phosphorylation.

Plasmid construction and transfection was done as described in Example1.

No significant fluorescence was observed from the nucleus of the probe(d)-expressing cells. It was confirmed that the probe for imagingprotein phosphorylation (d) was removed from the nucleus (FIG. 5A).

Further, upon stimulating the cells expressing probe (d) with 100 nMinsulin, in the same manner as in Example 2, a progressive decrease inthe cytosolic emission ratio was observed (FIG. 5A). No significantdifference was observed in the time course of the probe (d) response,even when compared with that of probe (b) (FIG. 4B).

FIG. 5B shows the response of probe (d) to differing concentrations ofinsulin in CHO-IR cells. The accumulation rate of phosphorylated probe(d) by insulin receptor was increased in parallel with increasinginsulin concentration. When the concentration of insulin was 0.1 nM, noaccumulation of phosphorylated probe (d) was observed.

The relation between the emission ratio of probe (d) and insulinconcentration was similar to the results reported for tyrosinephosphorylation of native IRS-1 protein in the cell, previously measuredby autoradiography (EMBO J. 16, 5573-5581 (1997)).

The above results indicate that probe (d) is suitable as a probe formulti-cell analysis that utilize fluorescence multi-well plate reader,wherein the probe protein in the cytosol and the nucleus cannot bediscriminated.

Hence, by using probe (d), high-throughput screening of anti-diabeticsmall molecules, such as L-783, 281 (Nature 318, 183-186 (1985); Science284, 974-977 (1999)), which were reported to directly stimulate thekinase activity of insulin receptor, from thousands of candidatechemicals, may be realized.

Example 4 Probe for Imaging Protein Phosphorylation andDephosphorylation Comprising a Living Cell Membrane Binding Sequence

Signal transduction proteins, such as kinases, phosphatases and theirsubstrates, are often localized in the cell and are organized to formdomains of signal transduction by extracellular stimuli. This mechanismis thought to be a critical factor to determine the efficiency andspecificity of signal transduction in the cell.

It has been known that IRS-1, which is the endogenous substrate proteinfor insulin receptor, contains a pleckstrin homology (PH) domain and aphosphotyrosine binding (PTB) domain to its N-terminal end (Diabetologia40, S2-S17 (1997)).

The PH and PTB domain bind with the phosphoinositides of the cellmembrane and with the juxtamembrane domain of insulin receptor, which istyrosine-phosphorylated by insulin simulation, respectively (Proc. Natl.Acad. Sci. USA 96, 8378-8383). Thus, the concentration of IRS-1 isincreased around insulin receptor at the plasma membrane upon insulinstimulation, which underlies efficient and selective phosphorylation ofIRS-1 by insulin receptor (J. Biol. Chem. 270, 11715-11718 (1995)).

Then, probe (b) was fused with PH-PTB domain derived from the IRS-1protein to construct the probe of FIG. 2( e).

CHO-IR cells expressing probe (e) were stimulated with 100 nM of insulinfor 7 min at 25° C. The cells were fixed with 2% paraformaldehyde andwere permeabilized with a phosphate-buffered saline containing 0.2%Triton X-100 for 10 min. After 45 min of incubation with rabbit anti-βsubunit of human insulin receptor antibody (1:100 dilution), the cellswere washed with a phosphate-buffered saline containing 0.2% fish skingelatin and incubated with anti-rabbit IgG antibody labeled with Cy5(1:500) for 30 min.

The coverslips were mounted onto the slide and observed with a confocallaser scanning microscope (LSM510, Carl Zeiss).

FIG. 6A shows a comparison of the cytosolic emission ratio change forprobe (e) and that for probe (b) in CHO-IR cells when stimulated with100 nM insulin. Although the rate of the cytosolic emission ratio changefor probe (e) was significantly faster than that for probe (b), bothemission ratios r were not significantly different when they plateaued.

Accordingly, this indicates that by introducing the endogenous targetingdomain within IRS-1, the phosphorylation rate of probe (e) by theactivated insulin receptor was enhanced, which demonstrated that thelocalized kinase signaling in the living cells can be visualizedeffectively using this probe for imaging protein phosphorylation.

Insulin-stimulated co-localization of probe (e) and the insulin receptorat the plasma membrane were confirmed by the fluorescence images takenby the confocal laser scanning microscope (FIG. 6B). This membranelocalization of probe (e) was not observed before insulin simulation. Onthe other hand, when probe (b) was used, no significant subcellularlocalization, including the plasma membrane, by insulin stimulation wasobserved (FIG. 4B).

From these results, it was demonstrated that the PH-PTB domain wasascribed to be responsible for the insulin-induced targeting of probe(e) to the membrane insulin receptor.

Furthermore, it is suggested that upon insulin stimulation, SH2n withinthe probe for imaging protein phosphorylation preferentially binds viaintramolecular reaction with the adjacent phosphorylated Y941 ratherthan binding via intermolecular reaction with the other localizedphosphoproteins such as endogenous IRS proteins (J. Biol. Chem. 273,29686-29692 (1998); Mol. Endocrinol. 14, 823-836 (2000))

INDUSTRIAL APPLICABILITY

As described above in detail, the present invention provides a methodfor imaging signal transduction caused by protein phosphorylation withinliving cells. The present invention not only enables the visualizationof kinase signal transduction within single live cells in high spatialand temporal resolution, but also enables the high-throughput screeningof substances that regulate the activity of various phosphorylation anddephosphorylation substances. Further, by generating transgenic animalsor plants using the probe for imaging protein phosphorylation of thepresent invention, a nondestructive continuous method for monitoringevents related to signal transduction due to protein phosphorylationwithin target tissues and organs can be realized.

1. A probe for imaging protein phosphorylation and dephosphorylation,which comprises: (a) a peptide substrate domain containing aphosphorylation site having an amino acid residue selected fromtyrosine, serine and threonine; (b) a phosphorylation recognition domainthat is a single chain antibody obtained using the phosphorylatedpeptide substrate domain as an antigen, or an endogenous domain selectedfrom SH2 domain, phosphotyrosine binding domain and WW domain, andspecifically interacts and recognizes phosphorylation of the peptidesubstrate domain; and (c) a peptide linker sequence, wherein: (i) thepeptide substrate domain and the phosphorylation recognition domain arebound together by the peptide linker sequence and interposed between adonor chromophore and an acceptor chromophore that cause fluorescenceresonance energy transfer (FRET) to occur; and (ii) the peptidesubstrate domain, the linker sequence and the phosphorylationrecognition domain are linked in order from N-terminal to C-terminal ofthe probe.
 2. The probe for imaging protein phosphorylation anddephosphorylation of claim 1, wherein the donor chromophore and theacceptor chromophore that cause FRET to occur, are fluorescent proteinseach having different fluorescence wavelengths.
 3. The probe for imagingprotein phosphorylation and dephosphorylation of claim 1, wherein thedonor chromophore and the acceptor chromophore that cause FRET to occur,are different color mutants of a green fluorescent protein.
 4. The probefor imaging protein phosphorylation and dephosphorylation of claim 3,wherein the mutants of the green fluorescent protein are a cyanfluorescent protein and a yellow fluorescent protein.
 5. The probe forimaging protein phosphorylation and dephosphorylation of claim 1, whichcomprises a localization sequence at a terminal end.
 6. The probe forimaging protein phosphorylation and dephosphorylation of claim 1,wherein in the probe: the donor chromophore and the acceptor chromophorethat cause FRET to occur are selected from a cyan fluorescent proteinand a yellow fluorescent protein, the linker sequence consists of theamino acid sequence of SEQ ID NO: 2, and the phosphorylation recognitiondomain is an N-terminal SH2 domain of p85 regulatory subunit of bovinephosphatidylinositol 3-kinase.
 7. A method for screening substances thatenhance or suppress protein phosphorylation, which comprises making theprobe for imaging protein phosphorylation and dephosphorylation of claim1 coexist with a candidate substance, measuring the change in efficiencyof fluorescence resonance energy transfer (FRET) before and afteraddition of the candidate substance, comparing the efficiency of FRETbefore and after addition of the candidate substance, and identifyingthe substance that enhances or suppresses protein phosphorylation. 8.The method for screening substances that enhance or suppress proteinphosphorylation of claim 7, wherein the probe for imaging proteinphosphorylation and dephosphorylation and the candidate substance aremade to coexist by introducing the probe for imaging proteinphosphorylation and dephosphorylation into cells.
 9. A method forscreening substances that enhance or suppress protein dephosphorylation,which comprises making the probe for imaging protein phosphorylation anddephosphorylation of claim 1, wherein the substrate domain has beenphosphorylated, coexist with a candidate substance, measuring the changein FRET efficiency before and after addition of the candidate substance,comparing the efficiency of FRET before and after addition of thecandidate substance, and identifying the substance that enhances orsuppresses protein dephosphorylation.
 10. The method for screeningsubstances that enhance or suppress protein dephosphorylation of claim9, wherein the probe for imaging protein phosphorylation anddephosphorylation and the candidate substance are made to coexist byintroducing the probe for imaging protein phosphorylation anddephosphorylation into cells.
 11. A method for assaying an amount of asubstance that enhances or suppresses protein phosphorylation, whichcomprises contacting the probe for imaging protein phosphorylation anddephosphorylation of claim 1 with various concentrations of a standardsubstance having a known enhancing or suppressing-ability to proteinphosphorylation, and measuring fluorescence resonance energy transfer(FRET) efficiencies thereof to produce a calibration curve as a control,introducing the probe into cells, introducing a candidate substance thatenhances or suppresses protein phosphorylation into the cells, measuringthe change in FRET efficiency before and after of the candidatesubstance upon contacting the probe with the candidate substance in thecells, and determining the amount of the candidate substance bycomparing the FRET efficiency of the candidate substance with thecalibration curve.
 12. A method for assaying an amount of a substancethat enhances or suppresses protein phosphorylation, which comprisescontacting the probe for imaging protein phosphorylation anddephosphorylation of claim 1 with various concentrations of thesubstance having a known enhancing or suppressing-ability to proteinphosphorylation, and measuring fluorescence resonance energy transfer(FRET) efficiencies thereof to produce a calibration curve as a control,introducing the probe into cells, introducing the substance into thecells, measuring the change in FRET efficiency before and after additionof the substance upon contacting the probe with the substance in thecells, and determining the amount of the substance by comparing the FRETefficiency of the substance with the calibration curve.